When cells are stimulated with a growth factor and so forth, information is transmitted inside the cells via receptors on the cell surface layer. In many cases, this transmission of information is known to be performed by phosphorylation of protein. Many receptors are protein kinases or form complexes with protein kinases. Information of growth factor etc. activates protein kinase in this manner resulting in phosphorylation of the target protein, which is then transmitted inside the cell so that the response of the cell begins.
Protein kinase is an enzyme that transfers the phosphoric acid at the .gamma.-position of ATP to a hydroxyl group of serine, threonine, tyrosine and so forth, and plays the role of a central mechanism in the transmission of information into cells. Protein kinases play an important role in the function regulatory mechanisms of almost all cells. For example, they have been clearly shown to be involved in cell movement, cell growth, metabolic response, immune response and so forth. In this way, in order to understand the function regulatory mechanisms of cells, it is essential to determine the target proteins of protein kinases. However, since the substrate proteins of protein kinases are not always clear, many unknown aspects remain regarding their details.
Efforts have been made in the past to explain the phosphorylation reaction in which protein kinases are involved. For example, Carmel, G. & Kuret, J. reported that the substrate selectivity of protein kinase was analyzed by expressing a DNA library containing a mutant gene fragment of a protein kinase substrate protein constructed using the cassette mutation induction method in an E. coli expression system, and performing a phosphorylation reaction in the solid phase in the presence of protein kinase (Analytical Biochemistry (1992), 203, 274-280). However, this method is limited to analysis of the site that recognizes the substrate protein on the protein kinase, and there is no mention of expression cloning of a gene that codes for protein kinase substrate protein. Thus, a method for efficiently cloning a gene that codes for protein kinase substrate protein was unknown.